Traditional basophilic kinases in the AGC family (like PKC and ROCK) are widely proposed to be the dominant ERM kinases, but the evidence is less than conclusive and, in particular, genetic evidence is lacking in vertebrates. This is of particular importance to NCI, because increasing evidence points to ERM phosphorylation as a process relevant to cancer metastasis. We exploited findings from our mass spectrometric analysis and identified a previously obscure kinase LOK (lymphocyte-oriented kinase) as a candidate based on its prominent enrichment in the membrane/microvillus fraction. Immunofluorescence studies demonstrate that LOK is appropriately localized at the plasma membrane to mediate ERM phosphorylation. Peptide specificity analysis shows it to be a basophilic kinase with a predicted optimum sequence resembling the ERM site, including an unusual preference for tyrosine at P 2. Protein phosphorylation analysis demonstrates phosphorylation of ERM that is at least comparable in rate to two favored candidate ERM kinases: PKC and ROCK. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have &gt;50% reduction in ERM phosphorylation. Taken together the findings on localization and specificity argue strongly that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations make sense in the context of current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of major importance in lymphocytes and elucidate a role for it in regulating cell shape and motility. We published these seminal findings on LOK this year in Proc Natl Acad Sci. However, ERM phosphorylation in the lymphocytes of LOK knockout mice is about 40% of normal, indicating that there are additional relevant kinases beyond LOK. The strongest candidate is SLK, which is the most similar to LOK in sequence and together LOK/SLK are the counterparts of the drosophila kinase (SLIK) which has been shown to mediate all ERM phosphorylation. Unlike LOK, SLK is widely expressed. Therefore, in planning generation of a knockout mouse to understand the in vivo function of SLK, who chose a conditional knockout design. We have designed and generated gene targeting constructs suitable for making conditional knockout mice, generated and screened ES cell clones and are now injecting ES cells to produce chimera. Concurrently, we are undertaking two complementary lines of further investigation of LOK. First, we have combined systematic analysis of sequence conservation in the kinase domain, and molecular modeling, to predict the structural basis of LOK peptide specificity. We are mutating those residues, and will analysis the peptide specificity of the recombinant protein. Second, LOK peptide specificity seems to have both a narrower specificity and less conformity to a PSSM-predicted motif than many basophilic kinases. Therefore we are exploring new powerful approaches for testing its specificity on proteomic peptides, under the hypothesis that there are more than one underlying patterns which can be better discriminated with additional information. As outlined in last years annual repot, we have developed a transgenic mouse model as important complementary approach to assessing the roles of ERM phosphorylation. The transgenic mouse expresses a mutant ezrin gene in which the C-terminal phosphorylation site is mutated to an acidic residue to mimic constitutive phosphorylation (T567E) and tagged with a short peptide HA tag at the extreme C-terminus. The gene is selectively expressed in T-cells (by use of a CD2 cassette) and protein expression is at most half of the endogenous ezrin levels. Studies of this mouse are ongoing and include comparisons with a transgenic mouse strain expressing a comparable construct without the mutation. We find a pattern of complex immune system abnormalities which is of modest severity in the heterozygote mouse, which are markedly more severe in a homozygous mouse (with higher level expression). The most notable alterations observed are reduced in vivo are impaired migration in lymph node, and dramatically impaired resistance to intra-peritoneal Toxoplasma infection. The biochemical basis for these alterations is being investigated. Fractionation studies confirm that the ezrin T567E protein is strongly enriched at the membrane in contrast to the wildtype transgenic protein. Calcium signaling in response to TCR cross-linking is substantially impaired. Moreover, some key aspects of cytokine signaling are altered. Moreover, cell survival is impaired in culture under some conditions. We are seeking to understand the molecular connections between these pleiotropic alternations.